Xylitol

Toxicity to reproduction

Administrative data

Endpoint:

three-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Principles of method if other than guideline:

A three-generation study was conducted in CD rats with 20 males and 20 females per group. Each group received the test material by dietary administration. Observations were made to determine litter size, litter and pup weights, pup mortality, and gross abnormalities. Gross and histopathological observations were conducted on rats of the F3b generation.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Xylitol

EC Number:

201-788-0

EC Name:

Xylitol

Cas Number:

87-99-0

Molecular formula:

C5H12O5

IUPAC Name:

(2R,3r,4S)-pentane-1,2,3,4,5-pentol

Details on test material:

- Purity: not reported

Test animals

Species:

rat

Strain:

other: CD

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Manston, Kent, England
- Age at study initiation: not reported
- Weight at study initiation: (F0) Males: 76 g; Females: 72 g; (F1) Males: 122 - 130 g; Females: 107 - 113 g; (F2) Males: 115 - 130 g; Females: 107 - 119 g
- Housing: During the pre-mating period the rats were housed five to a cage with cages of males interspersed among those holding females to promote the development of regular oestrus cycles. After mating males were returned to their original cages, whilst the females were transferred to individual cages (equipped with solid floors) for the birth and rearing of litters.

- Diet: ad libitum (Spratt's Laboratory Diet No. 2)
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 5%
- Air changes (per hr): 12 - 14

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION: The test compounds were administered in the diet (Spratt's Laboratory Diet No. 2) and fresh batches of diet were mixed weekly. Treatment continued throughout the study for all generations. Concentrations of greater than 5% were attained by weekly increments following a 5, I0 and 20% sequence to achieve appropriate concentrations with reciprocal reduction of rice starch.

Details on mating procedure:

- M/F ratio per cage: 1 to 1
- Length of cohabitation: 14 days
- Mating for second litter: Shortly following (approximately 10 days) sacrifice of the first litters, the animals were re-mated (for a period of 19 days) ensuring that different males and females were coupled within each group.
- Proof of pregnancy: Daily vaginal smears were taken during the mating period and the day on which spermatozoa were present (or a copulation plug found) was considered day 0 of pregnancy.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

A control group received 20% rice starch, and a comparison group 20% sucrose ad libitum.
Graded diets of test substance, 2, 5, 10 or 20% were fed to treatment groups with rice starch q.s. to bring the level to 20%. For test substance groups receiving diets greater than 5%, administration began at the 5% level with 5% weekly increments until final doses of 5, 10, 15 or 20% were reached.

During parturition, the diets were reduced to 5% test substance.
After parturition of second litters of each generation, dams receiving diets greater than 5% were returned to this level.

Males and non-pregnant females received full dose.
After weaning, the dose administered to F1B and F2B generations were progressively increased until full treatment level was attained.

Frequency of treatment:

Daily in feed

Details on study schedule:

- Selection of parents: Once animals of the F0 generation (and, subsequently, weanlings of the F1B and F2B generations) had been acclimatised to their full dietary concentrations they were maintained on their respective diets until females had attained 180/200 g weight (or were at least 9 weeks old). The animals were then mated on a one male to one female basis for a minimum period of 14 days, subsequently, males were returned to their original cages, whilst the females were transferred to individual cages (equipped with solid floors) for the birth and rearing of litters. Offspring of this pairing were sacrificed on day 4 post-partum.

Shortly following (approximately 10 days) sacrifice of the first litters, the animals were re-mated (for a period of 19 days) ensuring that different males and females were coupled within each group. In the first generation, all offspring of the second pairing were reared to 21 days pos- partum. However, in an attempt to overcome the high, but non-selective, pup mortality which occurred between days 4 and 12, agreement was reached with our sponsors to cull second litters of the subsequent F1B and F2B generations on day 4 post-partum to give an equalised litter size of eight, where possible.

From the second litters of the initial (F0) and second (F1B) generations, twenty males and twenty females were selected from each group at weaning to form the basis of the second and third (F2B) generations, respectively. The animals were selected from as many litters as practical, derived from the first two weeks' mating, and as close to the mean weaning weight for each sex as possible. Brother and sister matings were avoided for the second and third generations.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 other: %

Remarks:

nominal in diet

Doses / concentrations 2

Dose / conc.:

5 other: %

Remarks:

nominal in diet

Doses / concentrations 3

Dose / conc.:

10 other: %

Remarks:

nominal in diet

Doses / concentrations 4

Dose / conc.:

20 other: %

Remarks:

nominal in diet

No. of animals per sex per dose:

Twenty male and female rats from each dose group

Control animals:

yes, plain diet

Positive control:

Rats were tested with 20% Sorbitol and 20% Sucrose

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Any rat which showed marked signs of ill health or reaction to treatment was isolated and/or killed to prevent cannibalism or autolytic degeneration. All animals that died or were killed for humane reasons were weighed and subjected to post mortem examination.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat of the F0 generation was determined initially and subsequently at weekly intervals up to pairing. Animals of the F1B generation were weighed at birth, 4, 12 and 21 days post-partum and subsequently at intervals of one week to pairing. For the F2B generation, pups were weighed, additionally, on day 8 post-partum. During the mating periods, females were also weighed on alternate days until the occurrence of a positive indication of mating (i.e. sperm or plug): thereafter they were weighed on presumed days 0, 7, 14 and 20 of gestation. Weights of pregnant animals without positive indication of mating were calculated retrospectively from birth assuming a 22-day gestation period. Dams were weighed on days 0, 7, 14 and 21 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Yes; Food intake of rats was recorded weekly during the first pre-mating period of each generation (F0, F1B, F2B).

OTHER:
PREGNANCY RATE: Pregnancy rate was determined as the percentage of surviving paired females that became pregnant.

MATING PERFORMANCE: Daily vaginal smears were taken during the mating period and the day on which spermatozoa were present (or a copulation plug found) was considered day 0 of pregnancy.

Oestrous cyclicity (parental animals):

Oestrous cycle length and normality were evaluated in F0 and F1 females by vaginal smears prior to mating, and during mating until evidence of mating was found.

Litter observations:

LITTER OBSERVATIONS: As soon as possible after parturition, which was observed wherever possible, the young were counted (for numbers of live and dead), weighed individually and examined for external abnormalities. Keeping nest disturbance to a minimum, all litters were examined daily for dead and abnormal young.

First Litters (F1A, F2A, F3A):
Young of the F1A generation were weighed individually on day 4 post-partum and sex was determined by external inspection of the ano-genital distance. Young were then sacrificed and examined externally and internally for abnormalities; sex of the pups was confirmed by gonadal inspection. Where indicated, pups were preserved in the appropriate fixative for further examination. Young of the F2A and F3A generation were individually identified within the litter as soon as possible after birth and the generation subsequently proceeded as for F1A.

Second litters (F1B, F2B, F3B):
As soon as possible after birth, young of the F1B generation were individually identified within the litter (toe marked) and weighed on days 4, 12 and 21 post-partum. Young of the F2B and F3B generations were weighed and sexed at day 4 post-partum when the litter size was reduced to 4 males and 4 females (total 8) except for occasional instances where this was made impractical by the original constitution of the litter. Reduction of the litter size was achieved by selecting for continuation 4 males and 4 females as close to the mean weight of each sex in the litter, the surplus males and females were then killed and examined. Young were weighed on days 8, 12 and 21 post-partum. Surplus pups of the F1B and F2B generations were sacrificed on, or shortly after, day 21 and examined externally and internally for abnormalities; sex of the pups was confirmed by gonadal inspection.

Parameters examined/calculated:
- Litter size: at birth, mean values were determined both for the total number of young and for the number of viable young
- Litter weight: calculated from the individual pup weights
- Pup mortality: calculated as % losses within individual litters at birth and cumulatively on days 4, (8), 12, and 21 (for equalised litters of the F1A generation, cumulative loss was calculated from day 4). Group mean values were calculated from individual litter percentages
- Abnormalities: All dead young (except those excessively cannibalised) were autopsied. Pups with suspected abnormalities were preserved either in Bouin's solution if it was considered that free-hand sectioning (Wilson technique) or histopathological examination would be of value, or in alcohol for subsequent staining with alizarin (modified Dawson technique) if skeletal defects were suspected.

Postmortem examinations (parental animals):

After the second litters had been weaned, parent animals of each generation (F0, F1B, F2B) were sacrificed and examined macroscopically.

Postmortem examinations (offspring):

Rats of the F3B generation were killed at 3 weeks of age and subjected to detailed external and internal examination for detection of abnormal development as for previous generations.

See Table 1 below

Statistics:

Litter data were statistically analysed on a litter basis using non-parametric methods. Absolute and relative organ weights were subjected to analysis of variance. Analysis of covariance was also carried out adjusting for bodyweight. Williams test for comparing increasing doses of a compound with a control was used for Xylitol groups and also, independently, for Sorbitol and Sucrose against the control group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males fed the test substance at 20%, suppression of weight gain became discernible once full dietary concentrations were achieved. Bodyweight progressively diverged from the control group, resulting in markedly lower terminal weights. Amongst females, the pattern of bodyweight gain duplicated that of males although divergences from the control group were slightly less pronounced.

For dams which successfully reared young - either to day 4 post-partum (1st mating) or to weaning (2nd mating) - weight gain during gestation generally reflected the patterns established prior to mating. Considered overall, post-partum weight change of dams fed the test substance at 2, 5, or 10% showed no adverse association with treatment. Those at 20% compared favourably with all other groups in the parental generation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was slightly lower amongst males after the full dietary concentration of 20% had been attained.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

At each mating the mean duration of gestation (approximately 22 days) in all groups was within normal limits for controls and essentially comparable. However, subtle intergroup variations within the normal range were consistently recorded over 3 generations. At 20%, the proportion of litters born on day 23 or 24 was generally greater than among controls, and was generally greater at the first mating than at the second, etc.

Results: P1 (second parental generation)

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

20 other: % (highest dose tested)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

2 males (one at 2% and one at 20%) and 2 females (one control and one at 20%) died during the study. The deaths were considered coincidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males fed the test substance at 20%, suppression of weight gain became discernible once full dietary concentrations were achieved. Bodyweight progressively diverged from the control group, resulting in markedly lower terminal weights. Amongst females, the pattern of bodyweight gain duplicated that of males although divergences from the control group were slightly less pronounced.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Marked suppression of food intake was recorded amongst both males and females, even at the lowest level of inclusion (5%).

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

At each mating the mean duration of gestation (approximately 22 days) in all groups was within normal limits for controls and essentially comparable. However, subtle intergroup variations within the normal range were consistently recorded over 3 generations. At 20%, the proportion of litters born on day 23 or 24 was generally greater than among controls, and was generally greater at the first mating than at the second, etc.

Details on results (F1)

At the 20% dietary level of test substance there were lower values for total and viable litter size at birth and at day four postpartum. This became more accentuated with successive generations. Litter and mean pup weights were greater than concurrent control values except for 20% test substance at which level lower litter weights reflected smaller litter size. Initially among the F1a pups, when litter size was not culled, a large mortality was noted between seven to 10 days postpartum. The litter size was culled to eight with the succeeding generations, at which time no total litter loss was observed. There was no indication of a treatment effect on occurrence of terata.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

other: F1A and F1B

Effect level:

20 other: % (highest dose tested)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males fed the test substance at 20%, suprpession of weight gain became discernible once full dietary concentrations were achieved. Bodyweight progressively diverged from the control group, resulting in markedly lower terminal weights. Amongst females, the pattern of bodyweight gain duplicated that of males although divergences from the control group were slightly less pronounced.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Marked suppression of food intake was recorded amongst both males and females, even at the lowest level of inclusion (5%).

Gross pathological findings:

no effects observed

Description (incidence and severity):

During terminal sacrifice of F2B parents caecal enlargement appeared to be occurring in some groups and thereafter the weight of the caecum and contents of remaining dams and of ten males per group was determined. Although visual examination did not always indicate overt caecal enlargement, the weighing showed on increased caecal weight among Sorbitol fed animals and a dosage related increase in caecal weight for test substance fed animals, the effect being greater among females than males. Except for both sexes, fed 2% test substance and males fed 10% test substance intergroup differences from control values were statistically significant.

Histopathological findings:

no effects observed

Description (incidence and severity):

Histopathologically tissues from 10 and 20% test substance groups did not show microscopic changes that could be attributed to test substance.

Details on results (F2)

At the 20% dietary level of test substance there were lower values for total and viable litter size at birth and at day four postpartum. This became more accentuated with successive generations. Litter and mean pup weights were greater than concurrent control values except for 20% test substance at which level lower litter weights reflected smaller litter size. Initially among the F1a pups, when litter size was not culled, a large mortality was noted between seven to 10 days postpartum. The litter size was culled to eight with the succeeding generations, at which time no total litter loss was observed. There was no indication of a treatment effect on occurrence of terata.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

other: F2A and F2B

Effect level:

20 other: % (highest dose tested)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

With the sucrose group food intake was comparable with or slightly greater than that of the control group in all generations. At the lower levels, weight gain of males of the first generation (2, 5, or 10%) was comparable to the control group, slight suppression occurred at the 10% level during the second generation, and at all concentrations in the final generation (F_3b). At the highest level (20%) a larger proportion of litters were born on day 23 or 24 compared to controls, or sucrose. Caecal enlargement was noted at terminal necropsy of F_2b parents of both sexes for levels greater than 2%.

Applicant's summary and conclusion

Executive summary:

The effect of the test substance on growth and reproductive performance of the rat was assessed in a 3-generation reproduction study. Test diets were fed continuously to both sexes at concentrations of 0, 2, 5, 10, or 20% except that concentrations above 5% were achieved by gradual increments from a 5% inclusion in the case of the F0 generation; subsequently, for nursing dams the dietary concentration was reduced to 5% immediately after the birth of the second litters and full dietary concentrations for F1B and F2B generations were achieved by gradual increments from a 5% concentration in the immediate post weaning phase. For comparison, further groups of animals were fed diets containing 20% of either sucrose or sorbitol; final concentrations being achieved in a similar manner to that adopted for 20% xylitol. For each generation 2 litters were obtained, the first being killed and examined at day 4 post-partum, the second being killed and examined at day 21 post-partum (weaning). Additional organ weight analysis and histopathological examinations were made on selected offspring of the final (F3B) generation.

 

Among parent animals (F0, F1B, F2B) there were no clinical signs of reaction and no deaths attributable to treatment; mating performance and pregnancy rate were unaffected by any diet.

 

Food consumption and bodyweight gain of both sexes fed xylitol at dietary concentrations of 2, 5, or 10% showed no consistent differences from control values over the 3 generations, although slight reductions in food intake and bodyweight gain were recorded for males of the F1B generation. With dietary concentrations of 20% xylitol, food consumption was reduced, and bodyweight gain retarded for each generation; both sexes were affected, the divergence from control values being more noticeable among males. Even greater reductions in food consumption and bodyweight gain were recorded for the group fed sorbitol, with divergence from control values occasionally being detectable before the final dietary concentration of 20% was achieved. In contrast, food consumption and bodyweight gains of sucrose fed rats tended to be comparable with or greater than that of controls particularly in the early growth phase of each generation. Weight changes associated with pregnancy generally reflected the pre-mating pattern in groups fed xylitol and sorbitol diets; the lowest weight gains being recorded for animals fed 20% xylitol or sorbitol; the higher weight gains recorded for the same groups during lactation would be consistent with the lower absolute bodyweight at commencement of lactation. Conversely, with sucrose the slightly lower weight gains recorded during pregnancy and lactation possibly reflected the higher absolute pre-mating bodyweights.

 

The duration of gestation was within the normal range for all groups, but there was a consistent tendency for greater proportions of rats to show a slightly longer-than-average duration of gestation in the group fed 20% sorbitol and, to a slighter lesser extent, in the group fed 20% xylitol.

 

Terminal autopsy of adult and young F0 and F1B generation animals showed no immediately obvious changes attributable to any specific diet. Additional weighing of the caeca of tF2B generation adults, however, revealed a significant dosage related trend for increased caecal weight in xylitol-fed groups and a significantly increased caecal weight in the sorbitol fed groups. Caecal weight of sucrose fed animals was comparable with that of the concurrent controls.

 

Among the first litters of each generation, 20% xylitol and sorbitol diets were associated with a lower total and viable litter size at birth; further divergence from control values was evident at day 4 post-partum as a consequence of an increased perinatal mortality. Conversely, significantly higher mean pup weights at birth and 4 days post-partum were recorded for the 2 groups in part at least, reflecting an inverse relationship between litter size and mean pup weight. Over the 3 generations, litter size and mean pup weight with xylitol at 2, 5, and 10% and sucrose 20% tended to be comparable with or greater than control values.

 

Among second mating litters a similar pattern of lower litter size at birth and 4 days was again recorded for 20% xylitol or sorbitol-fed groups, but divergence from control values was much less marked and with xylitol differences were not statistically significant. There increased perinatal pup mortality noted in the first litters in these groups was not recorded in the second litters. As in the first litters mean pup weight in these 2 groups at birth and 4 days was again higher than corresponding control values. Bearing in mind the inter-relationship between litter size and mean pup weight, values for other groups were again comparable with, or greater than, those recorded for controls.

 

Subsequent to day 4 post-partum, in the F0 generation, a high pup mortality rate occurred between days 7 to 11 post-partum in all groups, including controls. This was considered to be due to the fact the higher-than-average litter sizes (range 13.7 to 15.0 compared with a laboratory average of 12.4 for controls) placed an excessive burden on nursing dams during the period of peak lactation demand. For subsequent F1B and F2B generations, litter size was reduced to 8 at day 4 post-partum; no total litter losses occurred in these generations (compared with 14/137 for the F0 generation), and pup mortality rates from day 4 to 21 were in the order of 2% compared with the 36-52% loss recorded during days 7 to 11 in the F0 generation. The higher mean pup weights at birth and day 4 frequently observed for groups fed 20% xylitol or sorbitol, and occasionally for other groups, tended to converge and become comparable with control values during the remainder of lactation due to a lower mean pup weight gain. This lower pup weight gain during the second half of the lactation period was particularly evident in the final (F2B) generation for groups fed 20% xylitol or sorbitol and resulted in significantly lower weaning weight (21 days post-partum) of the F3B offspring compared to that of controls. In all other groups in all generations and during the first 2 generations for groups fed 20% xylitol or sorbitol, values for litter size and mean pup weight at weaning were never significantly lower than concurrent control values.

 

Occasional structural abnormalities and anomalies recorded at post mortem examination of young of all generations were considered to be unrelated to treatment.

 

Among F3B young selected for additional, more detailed examination, statistically significant differences in absolute mean weights of a number of organs were generally considered to be consequent to the depression in overall bodyweight of this generation as the majority of differences failed to retain statistical significance after adjustment for bodyweight. Additionally, differences in absolute or relative organ weight were not conclusively associated with any specific macroscopic or microscopic pathological changes.